The overall goals of the proposed research are to gain further insights into the molecular mechanisms of various liver plasma membrane ion transport processes. Specifically, studies are aimed at gaining further insights into the amino acid composition, molecular biology and immunologic properties of the specific membrane polypeptides which mediate these transport mechanisms. Such knowledge will hopefully contribute to a better understanding of hepatic transport function both in health and during bile secretory failure (cholestasis). The bulk of the proposal describes the application of protein sequencing, molecular biologic and immunologic techniques in studies of a rat 50,000 Mr basolateral membrane polypeptide mediating hepatic Na+-dependent conjugated bile acid uptake. Polyclonal antibodies to this polypeptide raised in rabbits will be used both in physiologic studies and to immunoprecipitate functionally intact polypeptide for reconstitution experiments in liposomes. Partial sequencing of the NH2-terminus will be performed. This information will form the basis for the synthesis of an oligonucleotide probe with which to screen a cDNA library from rat liver for specific cDNAs encoding this polypeptide. Nucleotide sequencing of these cDNAs will then allow deduction of the full amino acid sequence of the transport polypeptide. DNA transfection experiments in fibroblasts and studies of the expression of polypeptide-specific mRNA during cholestasis will also be performed. Studies aimed at identifying the carrier polypeptide mediating the Na+- independent uptake of unconjugated bile acids are also described and are based on the demonstration of substrate-specific protection against inhibition by group-specific reagents. If these preliminary studies are successful, strategies similar to those used for the Na+-dependent conjugated bile acid transporter will be used for molecular biologic studies. Finally, domain-specific Ca++ transporters on the basolateral and canalicular membranes will be studied both enzymatically and with a combined molecular biologic and immunologic approach.